The long term objective of this application is to understand G-protein function and regulation in retinal rods and to understand light-induced rod membrane conductance changes in terms of the functions of rod gene product ensembles. The specific aims are: to understand the physiological significance and to identify the mechanisms of phosducin regulation of transducin activity; to understand the biochemical mechanisms that permit rod outer segment gene products to continuously function in harmony with the dictates of such second messengers as cAMP, cGMP, and Ca2+; and to understand the physical meaning of, and to identify the molecular ensembles that are responsible for, positive cooperative binding of transducin to rhodopsin. In terms of the overall design and methods, protein components will be isolated, purified, reconstituted into ensembles and functionally evaluated in order to pursue the specific aims and objectives. Biochemical or physical parameters that modify the extent of cooperative transducin interaction will be utilized to study the cooperative mechanism and its kinetic consequences. Site-directed antibodies and peptides and site-directed mutagenesis will be utilized to probe the nature of interactive sites that participate in the phosphorylation-dependent affinity of phosducin for G-protein subunits. Protein chemistry, molecular biology and chromatographic methods will also be utilized to probe protein/protein interactions between transducin and rhodopsin or transducin and phosducin. Amino acid sequencing, PCR and genebank search will be employed to identify the 29 kDa protein that co-purifies with phosducin. Protein biochemistry and gene sequencing techniques will be used to identify and characterize the calcium-activated adenylate cyclase of rod outer segments.